Project Summary Multiple large-scale epidemiological studies reveal the lack of etiological understanding in approximately 30% of patients across various types of liver diseases, including acute liver failure, chronic hepatitis, cirrhosis and hepatocellular carcinoma. Given an intrinsic progression mode among these diseases, there has long been suspected for the existence of additional hepatotropic viruses. Using a newly developed method, named template-dependent multiple displacement amplification (tdMDA) that eliminates amplification artifacts, Illumina sequencing and bioinformatics tools, we have performed a virome analysis in serum samples (0.5 mL per samples) from 10 patients with acute liver failure (ALF). These patients had no etiological factors identified in spite of exhaustive examination by the Acute Liver Failure Study Group (ALFSG), an ongoing NIH-sponsored large clinical trial. Our vriome analysis and subsequent experiments have identified a 536-bp unknown sequence, named Fragment M, which has no analogy to any nucleotide sequences in all databases currently available, including the NCBI reference genome database, the NCBI non-redundant nucleotide database and the datasets from the human microbiome project (HMP) and marine microbial genome and seawater shotgun sequencing. It contains a coding region with the motif similar to the conserved domains GdpP (c-di-AMP phosphodiesterase) and PRK14538 (putative bifunctional signaling protein/50S ribosomal protein L9), sharing approximately 39% amino acid identity with bacterial poly (A) polymerase. Fragment M was RNA in nature and solely detected in 15% of ALF patients with unknown etiology (3/20) but not in ALF patients with known etiologies (0/20) or healthy blood donors (0/20). Taken together, these exciting data support that Fragment M is a viral candidate in ALF patients with unknown etiology. Based on this discovery, the current proposal is going to explore its full genome characterization as well as evolutionary relatedness to known viruses (Aim 1). Causal link with ALF will be validated through a series of experiment (Aim 2), including hepatotropic estimation, infection dynamics and comparative detection rates of the candidate virus among three populations, ALF patients with (n=81) or without explicit etiologies (n=200) and healthy blood donors (n=81). Thus, the proposed studies have the best opportunity for the unveiling of the mystery in liver diseases, unrecognized viral pathogens beyond hepatitis A through E.